PROJECT SUMMARY The gastrointestinal tract is colonized by a diverse collection of commensal microbiota that occupy a symbiotic niche with the host. It is therefore in the best interest of the immune system to tolerate these microbes while still remaining vigilant against other pathogenic species of microbes. Indeed, failure of the immune system to accomplish this critical function is presumed to be a major underlying cause of inflammatory bowel diseases (IBD). While much has been learned about the mechanisms establishing immune tolerance to self antigens, very little is known about how tolerance towards gut commensal microbial antigens is mediated, if at all. Accumulating evidence implicates a major role for regulatory T cells (Treg) as a dominant form of tolerance within the gastrointestinal environment. However, a dearth of relevant experimental tools has limited investigation into how Tregs and other CD4+ T cells may contribute to immune tolerance to gut commensal antigens. We have recently developed powerful experimental systems involving peptide:MHC tetramer-based cell enrichment techniques that allow us to directly characterize rare populations of commensal microbial antigen-specific T cells in the natural endogenous repertoires of mice. These tools will enable us to investigate T cell tolerance to commensal microbes at an unprecedented level of physiological significance. Our long term goal is to determine the role that CD4+ T cells play in maintaining immune tolerance to commensal microbiota of the gastrointestinal tract, and how they may be manipulated for therapeutic benefit. We hypothesize that T cell tolerance to gut commensal microbial antigens is peripherally induced and restricted to the gut associated lymphoid tissue (GALT). We believe that the mechanisms of tolerance are reversible, providing the adaptive immune system with a high level of flexibility that is needed to fine-tune immune responsiveness to the complex and dynamic antigen environment of the gut. We will test this hypothesis in this exploratory study by 1) characterizing commensal antigen-specific CD4+ T cells in gut-associated lymphoid tissue (GALT) versus systemic lymphoid environments, 2) characterizing commensal antigen-specific CD4+ T cells during colitis, and 3) developing new tools to study commensal antigen-specific CD4+ T cells. The achievement of our aims will greatly improve our understanding of how T cell tolerance is maintained to commensal microbiota in the gut, and how we may ultimately exploit this information for therapeutic purposes.